CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification

Cell. 2019 Jan 10;176(1-2):254-267.e16. doi: 10.1016/j.cell.2018.11.052.


The ability to engineer natural proteins is pivotal to a future, pragmatic biology. CRISPR proteins have revolutionized genome modification, yet the CRISPR-Cas9 scaffold is not ideal for fusions or activation by cellular triggers. Here, we show that a topological rearrangement of Cas9 using circular permutation provides an advanced platform for RNA-guided genome modification and protection. Through systematic interrogation, we find that protein termini can be positioned adjacent to bound DNA, offering a straightforward mechanism for strategically fusing functional domains. Additionally, circular permutation enabled protease-sensing Cas9s (ProCas9s), a unique class of single-molecule effectors possessing programmable inputs and outputs. ProCas9s can sense a wide range of proteases, and we demonstrate that ProCas9 can orchestrate a cellular response to pathogen-associated protease activity. Together, these results provide a toolkit of safer and more efficient genome-modifying enzymes and molecular recorders for the advancement of precision genome engineering in research, agriculture, and biomedicine.

Keywords: CRISPR-Cas; Cas9-CP; ProCas9; circular permutation; fusion proteins; genome editing; protein engineering.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Associated Proteins / chemistry
  • CRISPR-Cas Systems / physiology*
  • Clustered Regularly Interspaced Short Palindromic Repeats / physiology*
  • DNA / chemistry
  • Gene Editing / methods*
  • Genome
  • Models, Molecular
  • RNA / chemistry
  • RNA, Guide, Kinetoplastida / genetics


  • CRISPR-Associated Proteins
  • RNA, Guide
  • RNA
  • DNA